The objective of this proposal is to develop novel approaches for efficient hepatocyte-directed delivery of nucleic acids. The proposal is based on our previous work demonstrating that DNA can be targeted specifically to hepatocytes using a DNA carrier containing an asialoglycoprotein that is recognized by receptors on the plasma membrane of hepatocytes. We will test the major hypothesis that natural proteins and peptide components can be incorporated into complexes to enhance intracellular delivery of DNA and expression of genes targeted to hepatocytes. The project proposes to use a modular design for the preparation of conjugates for DNA delivery. With this strategy, separate conjugates will be prepared which incorporate empty parvoviral capsids, individual capsid proteins, and a bacterial protein listeriolysin O, as endosomolytic agents to promote escape of targeted DNA from endosomal vesicles into the cytoplasm following internalization. Nuclear targeting sequences derived from SV-40 T-antigen will be incorporated into conjugates to direct efficient migration of DNA to the nucleus following escape from endosomes. These modules will be assessed first individually, and then in combination. In this way, optimal systems will be developed to deliver DNA in the form of genes to produce new gene expression. In addition to DNA as genes, antisense oligonucleotides will be delivered to inhibit endogenous gene expression. Pharmacological and genetic approaches to prolong the duration of targeted gene expression will be explored. Conditions that increase the likelihood of integration of the foreign genes into the host genome will be tested. The modular systems and protocols will be tested for efficiency and duration of effects on gene expression in vitro and in vivo. Finally, the function of the DNA targeted as optimal modular complexes will be evaluated in animal models of genetic disorders.